Repair of a Site-Specific DNA Cleavage: Old-School Lessons for Cas9-Mediated Gene Editing

Gallagher, Danielle N; Haber, James E.

doi:10.1021/acschembio.7b00760

Cited by 71 publications

(70 citation statements)

References 127 publications

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“…Here, the Cas9-nuclease is targeted to a specific genomic locus by a short single guide RNA (sgRNA) (Jinek et al, 2012). The Cas9-mediated introduction of a double strand break (DSB) at the genomic locus triggers non-homologous end joining, an error-prone cellular repair mechanism, that leads to the insertion and/or deletion (InDels) at the DSB (Gallagher and Haber, 2017).…”

Section: Genome Editing By the Electroporation Of Cas9 Guide Rna Compmentioning

confidence: 99%

Genetic knockdown and knockout approaches in Hydra

Lommel

Tursch

Rustarazo-Calvo

et al. 2017

Preprint

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Hydra is a member of the Cnidaria, an ancient phylum at the base of metazoan evolution and sister group to all bilaterian animals. The regeneration capacity of Hydra, mediated by its stem cell systems is unparalleled in the animal kingdom. The recent sequencing of the Hydra genome and that of other cnidarians has drawn new attention to this well-known model organism. In spite of this, the establishment of methods to manipulate gene expression in Hydra have remained a major challenge. Here we report a CRISPR-Cas9 based targeted mutation approach as well as an optimized, reproducible strategy for the delivery of siRNAs. Both approaches are based on a refined electroporation protocol for adult Hydra polyps. We demonstrate that these strategies provide reliable genetic interference with target gene expression, facilitating functional studies and genome editing in Hydra.

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Section: Genome Editing By the Electroporation Of Cas9 Guide Rna Compmentioning

confidence: 99%

Genetic knockdown and knockout approaches in Hydra

Lommel

Tursch

Rustarazo-Calvo

et al. 2017

Preprint

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“…Subsequently, DNA complementary to the template is synthesized prior to ligation of the resulting sequence to the free DNA end [27][28][29]. This repair pathway is Following the failure of alternative EJ pathways, and in the presence of a double-stranded DNA template bearing homology to the area of the DSB, homologous recombination (HR) pathways are activated [25]. HR occurs exclusively during the S and G2 phases of the cell cycle in proliferating cells, when intact sister chromatids are available for use as repair templates [22], reaching peak activity in mid-S phase [26].…”

Section: Dna Repair Pathwaysmentioning

confidence: 99%

“…Distinct from HR, single-strand template repair (SSTR) involves the annealing of a single-stranded DNA template with resected 3 overhangs at the DSB. However, the mechanism of this pathway requires further elucidation [25]. HR and SSTR can be co-opted for the introduction of small and large sequence variations at the nuclease target site, including exon/gene insertions.…”

Section: Dna Repair Pathwaysmentioning

confidence: 99%

Context-Dependent Strategies for Enhanced Genome Editing of Genodermatoses

March

Köcher

Koller

2020

Cells

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The skin provides direct protection to the human body from assault by the harsh external environment. The crucial function of this organ is significantly disrupted in genodermatoses patients. Genodermatoses comprise a heterogeneous group of largely monogenetic skin disorders, typically involving mutations in genes encoding structural proteins. Therapeutic options for this debilitating group of diseases, including epidermolysis bullosa, primarily consist of wound management. Genome editing approaches co-opt double-strand break repair pathways to introduce desired sequence alterations at specific loci. Rapid advances in genome editing technologies have the potential to propel novel genetic therapies into the clinic. However, the associated phenotypes of many mutations may be treated via several genome editing strategies. Therefore, for potential clinical applications, implementation of efficient approaches based upon mutation, gene and disease context is necessary. Here, we describe current genome editing approaches for the treatment of genodermatoses, along with a discussion of the optimal strategy for each genetic context, in order to achieve enhanced genome editing approaches.

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“…Nearby matching sequences are used as substrate for microhomology-mediated repair more frequently than distant ones. Fraction of mutated reads (y-axis) for increasing distance between matching sequences of length 9 (x-axis) for individual targets (blue markers) in K562 cells, and a linear regression fit to the trend (solid line Mutational outcomes vary with cell type and some Cas9 modifications Cells can differ in activity of repair processes and/or DNA sequence, both of which influence double strand break repair outcomes (Gallagher and Haber 2018) . To test whether these factors have an effect in the context of our analyses, we next performed our assay in human induced pluripotent stem cells (iPSCs), mouse embryonic stem cells (mESCs), Chinese hamster ovary (CHO) epithelial cells, as well as human retina epithelial immortalized cells (RPE-1) and leukemic near-haploid (HAP1) cell lines.…”

Section: Repair Outcomes Depend On Local Sequence Propertiesmentioning

confidence: 99%

Mutations generated by repair of Cas9-induced double strand breaks are predictable from surrounding sequence

Allen

Crepaldi

Alsinet

et al. 2018

Preprint

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The exact DNA mutation produced by cellular repair of a CRISPR/Cas9-generated double strand break determines its phenotypic effect. It is known that the mutational outcomes are not random, and depend on DNA sequence at the targeted location. Here, we present a systematic study of this link. We created a high throughput assay to directly measure the edits generated by over 40,000 guide RNAs, and applied it in a range of genetic backgrounds and for alternative CRISPR/Cas9 reagents. In total, we gathered data for over 1,000,000,000 mutational outcomes in synthetic constructs, which mirror those at endogenous loci. The majority of reproducible mutations are insertions of a single base, short deletions, or long microhomology-mediated deletions. gRNAs have a cell-line dependent preference for particular outcomes, especially favouring single base insertions and microhomology-mediated deletions. We uncover sequence determinants of the produced mutations at individual loci, and use these to derive a predictor of Cas9 editing outcomes with accuracy close to the theoretical maximum. This improved understanding of sequence repair allows better design of editing experiments, and may lead to future therapeutic applications.

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Repair of a Site-Specific DNA Cleavage: Old-School Lessons for Cas9-Mediated Gene Editing

Cited by 71 publications

References 127 publications

Genetic knockdown and knockout approaches in Hydra

Genetic knockdown and knockout approaches in Hydra

Context-Dependent Strategies for Enhanced Genome Editing of Genodermatoses

Mutations generated by repair of Cas9-induced double strand breaks are predictable from surrounding sequence

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